Mda-7/IL-24 exemplifies the ideal cancer gene therapeutic. It induces apoptosis selectively in diverse cancer cells without exerting deleterious effects to their normal counterparts, inhibits angiogenesis, stimulates an anti-tumor immune response, sensitizes cancer cells to other conventional modalities of therapy and exhibits profound 'bystander' anti-tumor activity eliminating both primary and distant tumors in animal models. Moreover, a replication incompetent adenovirus expressing mda-7/IL-24, Ad.mda-7, has been evaluated in a Phase I clinical trial for multiple solid tumors and melanomas demonstrating safety and significant objective anti-tumor clinical response. However, despite these intriguing properties, the mechanism(s) underlying the selective activity of this novel cytokine toward cancer cells remains to be defined. mda-7/IL-24 exhibits two modes of killing. As a secreted cytokine it binds to its cognate receptors and mediates anti-angiogenic, radiosensitization, immunostimulatory and 'bystander' activities. Alternatively, MDA-7/IL-24 protein, delivered by Ad.mda-7, localizes to the endoplasmic reticulum, interacts with the ER chaperone protein GRP78/BiP and generates an ER stress response. Additionally, Ad.mda-7 generates reactive oxygen species (ROS) in mitochondria and activates an apoptotic signaling cascade and also generates ceramide, a known inducer of apoptosis. All these phenomena, ER stress response, ROS and ceramide generation, are observed in the same human prostate cancer cells. However, these fragmented observations have not been linked by a unified molecular mechanism of action. The mechanism of 'bystander' activity also remains to be elucidated. Preliminary studies reveal that recombinant MDA-7/IL-24 protein induces expression of endogenous mda-7/IL-24. Accumulation of intracellular MDA-7/IL-24 might be a potential molecular mechanism for the 'bystander' effect providing a convergence between extracellular and intracellular tumor-specific apoptotic activities of this cytokine. We propose an in-depth analysis of molecular pathways of mda-7/IL-24 action, including its 'bystander' activity, to provide better insight into how this pleiotrophic tumor suppressor protein works. The information garnered will facilitate development of strategies to augment the anti-tumor effects of mda-7/IL-24. Considering the fact that Ad.mda-7 is already showing promise in clinical studies, successful completion of the proposed studies will significantly augment the therapeutic applications of mda-7/IL-24 potentially leading to elimination of both primary and metastatic tumors. PUBLIC HEALTH RELEVANCE: Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel secreted cytokine belonging to the IL-10 gene family that has the unique property of selectively inducing apoptosis in diverse cancer cells without harming normal cells or tissues. Additionally, secreted MDA-7/IL-24 protein generates a profound 'bystander' anti-tumor activity significantly enhancing its therapeutic properties. The present proposal aims at elucidating the molecular mechanism by which mda-7/IL-24 exerts its pleiotrophic effects as a cancer therapeutic. The studies we propose will provide valuable information that can be used to define rational approaches for enhancing the cancer therapy applications of mda-7/IL-24.